Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith

ABSTRACT

The invention provides reagents and methods for conjugating a polymer specifically to the α-amine of a polypeptide. The invention provides monofunctional, bifunctional, and multifunctional PEGs and related polymers having a terminal thioester moiety capable of specifically conjugating to the α-amine of a polypeptide having a cysteine or histidine residue at the N-terminus. The invention provides reactive thioester-terminated PEG polymers that have suitable reactivity with an N-terminal cysteine or histidine residue of a polypeptide to produce an amide bond between the PEG molecule and the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/973,318, filed Oct. 9, 2001, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to water soluble polymers useful forselectively conjugating to the N-terminus of a polypeptide.

BACKGROUND OF THE INVENTION

[0003] Covalent attachment of the hydrophilic polymer poly(ethyleneglycol), abbreviated PEG, also known as poly(ethylene oxide),abbreviated PEO, to molecules and surfaces is of considerable utility inbiotechnology and medicine. PEG is a polymer having the beneficialproperties of solubility in water and in many organic solvents, lack oftoxicity, and lack of immunogenicity. One use of PEG is to covalentlyattach the polymer to water-insoluble molecules to improve thesolubility of the resulting PEG-molecule conjugate. For example, it hasbeen shown that the water-insoluble drug paclitaxel, when coupled toPEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336(1995). PEG has also been used increasingly in the modification ofpolypeptide and protein therapeutics.

[0004] The use of polypeptides, including proteins, for therapeuticapplications has expanded in recent years mainly due to both improvedmethods for recombinant expression of human polypeptides from variousexpression systems and improved methods of delivery in vivo. Many of thedrawbacks associated with polypeptide therapeutics, including shortcirculating half-life, immunogenicity and proteolytic degradation, havebeen improved by various approaches including gene therapy, epitopemutations by directed or shuffling mutagenesis, shielding of the epitoperegions by natural or synthetic polymers, fusion proteins, andincorporation of the polypeptide into drug delivery vehicles forprotection and slow release.

[0005] Polymer modification of proteins, such as covalent attachment ofpoly(ethylene glycol), has gained popularity as a method to improve thepharmacological and biological properties of therapeutically usefulproteins. For example, certain poly(ethylene glycol) conjugated proteinshave been shown to have significantly enhanced plasma half-life, reducedantigenicity and immunogenicity, increased solubility and decreasedproteolytic degradation when compared to their non-pegylatedcounterparts. Factors that affect the foregoing properties are numerousand include the nature of the protein itself, the number ofpoly(ethylene glycol) or other polymer chains attached to the protein,the molecular weight and structure of the polymer chains attached to theprotein, the chemistries (i.e., the particular linkers) used to attachthe polymer to the protein, and the location of the polymermodified-sites on the protein.

[0006] To couple PEG to a molecule, such as a protein, it is oftennecessary to “activate” the PEG by preparing a derivative of the PEGhaving a functional group at a terminus thereof. The functional group ischosen based on the type of available reactive group on the moleculethat will be coupled to the PEG. For example, the functional group couldbe chosen to react with an amino group on a protein in order to form aPEG-protein conjugate.

[0007] A variety of methods have been developed to non-specifically orrandomly attach poly(ethylene glycol) to proteins. Most commonly,electrophilically-activated poly(ethylene glycol) is reacted withnucleophilic side chains found of proteins. Attaching an activatedpoly(ethylene glycol) to the α-amine and ε-amine groups found on lysineresidues and at the N-terminus results in a mixture of conjugateproducts as described in U.S. Pat. No. 6,057,292. For example, theconjugate may consist of a population of conjugated proteins havingvarying numbers of poly(ethylene glycol) molecules attached to theprotein molecule (“PEGmers”), ranging from zero to the number of α- andε-amine groups in the protein. Often, random pegylation approaches areundesirable, due to variations in the ratios of PEG-mer productsproduced, and the desire, in certain cases, for a single, discretePEG-protein conjugate product. For a protein molecule that has beensingly modified by employing a non-site specific pegylation methodology,the poly(ethylene glycol) moiety may be attached at any one of a numberof different amine sites. Additionally, this type of non-specificPEGylation can result in partial or complete loss of the therapeuticutility of the conjugated protein, particularly for conjugates havingmore than one PEG attached to the protein.

[0008] Several methods for site-directed or selective attachment of PEGhave been described. For example, WO 99/45026 suggests chemicalmodification of a N-terminal serine residue to form an aldehydefunctionality suitable for reaction with a polymer terminated with ahydrazide or semicarbazide functionality. U.S. Pat. Nos. 5,824,784 and5,985,265 suggest reacting a polymer bearing a carbonyl group with theamino terminus of a protein under reducing alkylation conditions and ata pH that promotes selective attack at the N-terminus. WO 99/03887 andU.S. Pat. Nos. 5,206,344 and 5,766,897 relate to the site-directedPEGylation of cysteine residues that have been engineered into the aminoacid sequence of proteins (cysteine-added variants). While these methodsoffer some advantages over non-specific attachment, there is acontinuing unmet need for improved methods and reagents for providingsite-specific polymer-conjugated proteins that do not require chemicalmodification of the polypeptide or careful control of certain reactionconditions, such as pH. Additionally, due to the high desirability formodifying a protein at its reactive amino-functionalities, there is aneed for improved polymer reagents that react selectively with aspecific protein amino group, such as the N-terminal amino group, forpreparing protein-polymer conjugates that are not a mixture ofPEG-polymer PEGmers but rather have PEG attached to a single, identifiedsite on the protein.

SUMMARY OF THE INVENTION

[0009] This invention provides reagents and methods for conjugatingpolymers specifically to the α-amino group of a polypeptide. Theinvention provides monofunctional, bifunctional, and multifunctionalPEGs and related polymers having a thioester (also referred to as athiol ester) moiety capable of specifically conjugating to the a-amineof a polypeptide having a cysteine or histidine at the N-terminus. Thus,the invention provides reactive thioester-terminated PEG polymerseffective to react site-specifically with an N-terminal cysteine orhistidine residue of a polypeptide to produce an amide-linkedPEG-polypeptide conjugate.

[0010] In one aspect, the invention provides a thioester-terminatedreactive polymer comprising a water soluble and non-peptidic polymerbackbone having at least one terminus bonded to the structure:

[0011] wherein,

[0012] L is the point of bonding to a water soluble and non-peptidicpolymer backbone;

[0013] Z is a hydrolytically stable linkage or a hydrolytically unstablelinkage, such as O, S, —NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

[0014] a is 0 or 1;

[0015] each X is independently selected from H and alkyl, such as C1-C6alkyl;

[0016] m is from 0 to about 12, preferably 1 to about 4;

[0017] Y is a heteroatom, preferably O or S; and

[0018] Q is a sulfur-containing leaving group preferably having theformula —S—R₁, wherein R₁ is hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocycle, or substituted heterocycle.

[0019] The reactive polymer may be monofunctional (e.g., mPEG),bifunctional, or multifunctional. The polymer backbone is preferably apoly(alkylene glycol), such as poly(ethylene glycol), poly(propyleneglycol), or a copolymer of ethylene glycol and propylene glycol.Examples of other suitable polymer backbones include poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), polyacrylate, polyacrylamides,polysaccharides, and copolymers, terpolymers, and mixtures thereof.

[0020] In another aspect, the invention provides a polymer conjugate ofa polypeptide having a cysteine or histidine molecule at the N-terminus,the polymer conjugate comprising a water soluble and non-peptidicpolymer backbone having at least one terminus bonded to the structure:

[0021] wherein

[0022] L, Z, m, Y, X and a are as defined above,

[0023] W is —CH₂SH or

[0024]  and

[0025] POLYPEPTIDE is a polypeptide molecule, where —NH—C(W)H—represents the N-terminal cysteine or histidine residue (absent onehydrogen atom) of the polypeptide. Examples of polypeptides that can beconjugated to the thioester-terminated polymers of the inventioninclude, but are not limited to, proteins, protein-ligands, enzymes,cytokines, hematopoietins, growth factors, hormones, antigens,antibodies, antibody fragments, receptors, and protein fragments.

[0026] In yet another aspect, a method of conjugating a polymerderivative to a polypeptide having a cysteine or histidine molecule atthe N-terminus is also provided. The method comprises providing both apolypeptide having a cysteine or histidine molecule at the N-terminusand a thioester-terminated polymer as described above. The polypeptideis reacted with the thioester-terminated polymer to form, in a sitespecific manner, a conjugate having an amide linkage between the residueof the N-terminal histidine or cysteine molecule and the reactivepolymer. The thioester-terminated polymer selectively attaches to theN-terminal amine group of the histidine or cysteine residue of thepolypeptide without reacting with free amine groups at other positionswithin the polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention now will be described more fullyhereinafter. This invention may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

[0028] I. Definitions

[0029] The following terms as used herein have the meanings indicated.

[0030] As used in the specification, and in the appended claims, thesingular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

[0031] The terms “functional group”, “active moiety”, “reactive site”,“chemically reactive group” and “chemically reactive moiety” are used inthe art and herein to refer to distinct, definable portions or units ofa molecule. The terms are somewhat synonymous in the chemical arts andare used herein to indicate the portions of molecules that perform somefunction or activity and are reactive with other molecules. The term“active,” when used in conjunction with functional groups, is intendedto include those functional groups that react readily with electrophilicor nucleophilic groups on other molecules, in contrast to those groupsthat require strong catalysts or highly impractical reaction conditionsin order to react (i.e., “non-reactive” or “inert” groups). For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Exemplary active esters include N-hydroxysuccinimidyl esters or1-benzotriazolyl esters. Typically, an active ester will react with anamine in aqueous medium in a matter of minutes, whereas certain esters,such as methyl or ethyl esters, require a strong catalyst in order toreact with a nucleophilic group. As used herein, the term “functionalgroup” includes protected functional groups.

[0032] The term “protected functional group” or “protecting group” or“protective group” refers to the presence of a moiety (i.e., theprotecting group) that prevents or blocks reaction of a particularchemically reactive functional group in a molecule under certainreaction conditions. The protecting group will vary depending upon thetype of chemically reactive group being protected as well as thereaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule, if any. Protecting groupsknown in the art can be found in Greene, T. W., et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y.(1999).

[0033] The term “linkage” or “linker” (L) is used herein to refer to anatom or a collection of atoms used to link, preferably by one or morecovalent bonds, interconnecting moieties such two polymer segments or aterminus of a polymer and a reactive functional group present on abioactive agent, such as a polypeptide. A linker of the invention may behydrolytically stable or may include a physiologically hydrolyzable orenzymatically degradable linkage

[0034] A “physiologically hydrolyzable” or “hydrolytically degradable”bond is a weak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. Preferred are bonds that have a hydrolysishalf life at pH 8, 25° C. of less than about 30 minutes. The tendency ofa bond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Appropriate hydrolytically unstable ordegradable linkages include but are not limited to carboxylate ester,phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, orthoesters, peptides and oligonucleotides.

[0035] A “hydrolytically stable” linkage or bond refers to a chemicalbond, typically a covalent bond, that is substantially stable in water,that is to say, does not undergo hydrolysis under physiologicalconditions to any appreciable extent over an extended period of time.Examples of hydrolytically stable linkages include but are not limitedto the following: carbon-carbon bonds (e.g., in aliphatic chains),ethers, amides, urethanes, and the like. Generally, a hydrolyticallystable linkage is one that exhibits a rate of hydrolysis of less thanabout 1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

[0036] An “enzymatically unstable” or degradable linkage is a linkagethat can be degraded by one or more enzymes.

[0037] The term “polymer backbone” refers to the covalently bonded chainof repeating monomer units that form the polymer. For example, thepolymer backbone of PEG is

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

[0038] where n typically ranges from about 2 to about 4000. As would beunderstood, the polymer backbone may be covalently attached to terminalfunctional groups or pendant functionalized side chains spaced along thepolymer backbone.

[0039] The term “reactive polymer” refers to a polymer bearing at leastone reactive functional group.

[0040] Unless otherwise noted, molecular weight is expressed herein asnumber average molecular weight (M_(n)), which is defined as$\frac{\sum{NiMi}}{\sum{Ni}},$

[0041] wherein Ni is the number of polymer molecules (or the number ofmoles of those molecules) having molecular weight Mi.

[0042] The term “alkyl” refers to hydrocarbon chains typically rangingfrom about 1 to about 12 carbon atoms in length, preferably 1 to about 6atoms, and includes straight and branched chains. The hydrocarbon chainsmay be saturated or unsaturated.

[0043] “Cycloalkyl” refers to a saturated or unsaturated cyclichydrocarbon chain, including bridged, fused, or spiro cyclic compounds,preferably comprising 3 to about 12 carbon atoms, more preferably 3 toabout 8.

[0044] The term “substituted alkyl” or “substituted cycloalkyl” refersto an alkyl or cycloalkyl group substituted with one or morenon-interfering substituents, such as, but not limited to, C3-C8cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; acetylene;cyano; alkoxy, e.g., methoxy, ethoxy, and the like; lower alkanoyloxy,e.g., acetoxy; hydroxy; carboxyl; amino; lower alkylamino, e.g.,methylamino; ketone; halo, e.g. chloro or bromo; phenyl; substitutedphenyl, and the like.

[0045] “Alkoxy” refers to an —O—R group, wherein R is alkyl orsubstituted alkyl, preferably C1-C6 alkyl (e.g., methoxy or ethoxy).

[0046] “Aryl” means one or more aromatic rings, each of 5 or 6 corecarbon atoms. Multiple aryl rings may be fused, as in naphthyl orunfused, as in biphenyl. Aryl rings may also be fused or unfused withone or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings.

[0047] “Substituted aryl” is aryl having one or more non-interferinggroups as substituents. For substitutions on a phenyl ring, thesubstituents may be in any orientation (i.e., ortho, meta or para).

[0048] “Heteroaryl” is an aryl group containing from one to fourheteroatoms, preferably N, O, or S, or a combination thereof, whichheteroaryl group is optionally substituted at carbon or nitrogen atom(s)with C1-6 alkyl, —CF₃, phenyl, benzyl, or thienyl, or a carbon atom inthe heteroaryl group together with an oxygen atom form a carbonyl group,or which heteroaryl group is optionally fused with a phenyl ring.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. Heteroaryl includes, but is notlimited to, 5-membered heteroaryls having one hetero atom (e.g.,thiophenes, pyrroles, furans); 5-membered heteroaryls having twoheteroatoms in 1,2 or 1,3 positions (e.g., oxazoles, pyrazoles,imidazoles, thiazoles, purines); 5-membered heteroaryls having threeheteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroarylshaving 3 heteroatoms; 6-membered heteroaryls with one heteroatom (e.g.,pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heteroaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

[0049] “Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

[0050] “Heterocycle” or “heterocyclic” means one or more rings of 5-12atoms, preferably 5-7 atoms, with or without unsaturation or aromaticcharacter and at least one ring atom which is not carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen. Multiple rings may befused, as in quinoline or benzofuran.

[0051] “Substituted heterocycle” is heterocycle having one or more sidechains formed from non-interfering substituents.

[0052] “Non-interfering substituents are those groups that, when presentin a molecule, are typically non-reactive with other functional groupscontained within the molecule.

[0053] Suitable non-interfering substituents or radicals include, butare not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl,C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10 cycloalkyl, C3-C10cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl,C2-C12 alkoxyalkyl, C7-C12 alkoxyaryl, C7-C12 aryloxyalkyl, C6-C12oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, —(CH₂)_(m)—O—(C1-C10alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substitutedalkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclicradical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C1-C10 alkyl), —C(O)—(C1-C10alkyl), C2-C10 thioalkyl, —C(O)O—(C1-C10 alkyl), —OH, —SO₂, ═S, —COOH,—NR, carbonyl, —C(O)—(C1-C10 alkyl)—CF₃, —C(O)—CF₃, —C(O)NR₂, —(C1-C10alkyl)—S—(C6-C12 aryl), —C(O)—(C6-C 12 aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C1-C10 alkyl) wherein each m is from 1 to 8,—C(O)NR, —C(S)NR, —SO₂NR, —NRC(O)NR, —NRC(S)NR, salts thereof, and thelike. Each R as used herein is H, alkyl or substituted alkyl, aryl orsubstituted aryl, aralkyl, or alkaryl.

[0054] “Heteroatom” means any non-carbon atom in a hydrocarbon analogcompound. Examples include oxygen, sulfur, nitrogen, phosphorus,arsenic, silicon, selenium, tellurium, tin, and boron.

[0055] The term “drug”, “biologically active molecule”, “biologicallyactive moiety” or “biologically active agent”, when used herein meansany substance which can affect any physical or biochemical properties ofa biological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular, as used herein,biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like. “Polyolefinic alcohol”refers to a polymer comprising a polyolefin backbone, such aspolyethylene, having multiple pendant hydroxyl groups attached to thepolymer backbone. An exemplary polyolefinic alcohol is polyvinylalcohol.

[0056] As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer backbonemay include a minor number of peptide linkages spaced along the lengthof the backbone, such as, for example, no more than about 1 peptidelinkage per about 50 monomer units.

[0057] “Polypeptide” or “poly(amino acid)” refers to any moleculecomprising a series of amino acid residues, typically at least about10-20 residues, linked through amide linkages (also referred to aspeptide linkages) along the alpha carbon backbone. While in some casesthe terms may be used synonymously herein, a polypeptide is a peptidetypically having a molecular weight up to about 10,000 Da, whilepeptides having a molecular weight above that are commonly referred toas proteins. Modifications of the peptide side chains may be present,along with glycosylations, hydroxylations, and the like. Additionally,other non-peptidic molecules, including lipids and small drug molecules,may be attached to the polypeptide. The polypeptide may comprise anycombination or sequence of amino acid residues. The polymers of theinvention are suitable for covalent attachment to both polypeptides andproteins.

[0058] “Amino acid” refers to organic acids containing both a basicamine group and an acidic carboxyl group. The term encompasses essentialand non-essential amino acids and both naturally occurring and syntheticor modified amino acids. The most common amino acids are listed hereinby either their full name or by the three letter or single letterabbreviations: Glycine (Gly, G), Alanine (Ala, A), Valine (Val, V),Leucine (Leu, L), Isoleucine (Ile, I), Methionine (Met, M), Proline(Pro, P), Phenylalanine (Phe, F), Tryptophan (Trp, W), Serine (Ser, S),Threonine (Thr, T), Asparagine (Asn, N), Glutamine (Gln, Q), Tyrosine,(Tyr, Y), Cysteine (Cys, C), Lysine (Lys, K), Arginine (Arg, R),Histidine (His, H), Aspartic Acid (Asp, D), and Glutamic acid (Glu, E).

[0059] By “residue” is meant the portion of a molecule remaining afterreaction with one or more molecules. For example, an amino acid residuein a polypeptide chain is the portion of an amino acid remaining afterforming peptide linkages with adjacent amino acid residues. “Oligomer”refers to short monomer chains comprising 2 to about 10 monomer units,preferably 2 to about 5 monomer units.

[0060] The term “conjugate” is intended to refer to the entity formed asa result of covalent attachment of a molecule, such as a biologicallyactive molecule, to a reactive polymer molecule, preferablypoly(ethylene glycol).

[0061] The term “leaving group” refers to an atom or collection of atomscovalently attached to an atom (such as a carbon atom) and that can bereadily displaced from the atom, taking with it its bonding electrons.Typically, the leaving group is an anion or a neutral molecule. Thebetter the leaving group, the more likely it is to depart from the atomto which it is bonded. Representative good leaving groups are those thatare the conjugate base of a strong acid.

[0062] “Multifunctional” in the context of a polymer of the inventionmeans a polymer having 3 or more functional groups attached thereto,where the functional groups may be the same or different.Multifunctional polymers of the invention will typically comprise fromabout 3-100 functional groups, or from 3-50 functional groups, or from3-25 functional groups, or from 3-15 functional groups, or from 3 to 10functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functionalgroups attached to the polymer backbone.

[0063] II. Thioester Polymers

[0064] In one aspect, the present invention providesthioester-terminated water soluble polymers capable of selectivelyreacting with the N-terminal amino group of a polypeptide to form apolymer-polypeptide conjugate comprising a single water soluble polymerchain attached at the N-terminus. Such a polymer-polypeptide conjugateis referred to herein as mono-substituted (meaning a polymer chain issubstituted at only one site of the polypeptide). Modification of apolypeptide at only a single site is beneficial because the likelihoodof a significant reduction in bioactivity due to the presence of thepolymer chain is lessened as compared to indiscriminate or randompolymer attachment at various and multiple sites along the polypeptidechain. Moreover, the polymers and method provided herein for formingsite-specific conjugates provide an additional advantage over commonlyemployed prior art methods since multiple protection/deprotection stepsto prevent reaction of the polymer with other reactive groups/positionscontained within the polypeptide are unnecessary. Additionally, suchsite selective modification eliminates the need for additional conjugatepurification steps to isolate particular (e.g., monopegylated) conjugatespecies. Thus, use of the thioester polymers of the invention can offerthe above advantages while additionally providing the beneficialproperties of water-soluble polymer attachment, such as increased watersolubility, enhanced plasma half-life, and decrease in proteolyticdegradation as compared to an unmodified polypeptide.

[0065] As explained in greater detail below, the thioester-terminatedpolymers of the invention selectively react with an N-terminal cysteineor histidine residue of a polypeptide. Without being bound by theory,the reaction involves nucleophilic attack of the thioester group byeither the thiol side chain of a cysteine residue or the imidazole sidechain of a histidine residue to form a thioester intermediate. Thethioester intermediate then undergoes a rapid rearrangement that resultsin transfer of the acyl group of the polymer to the terminal amine groupof the polypeptide, thereby producing a peptide bond between the polymerand the N-terminus of the polypeptide. As would be understood, sinceonly an N-terminal cysteine or histidine residue would provide the sidechain necessary for the initial reaction step (e.g., attack on thepolymer thioester carbonyl carbon by a reactive thiol group of a proteinhaving an N-terminal cysteine), the polymers of the invention will, viaa molecular rearrangement, specifically attach to the N-terminal aminewithout reacting with any other side chain amine groups that may bepresent in the polypeptide molecule. The present invention isparticularly useful for site-specific PEG attachment of polypeptidescontaining more than one free cysteine or histidine, even in theunfolded state. The polymers and conjugation methods of the presentinvention can be used to assist insoluble polypeptides that are in theunfolded state to refold to their native conformation.

[0066] The thioester-terminated polymers of the invention comprise apolymer backbone attached to a thioester group with an optionalintervening linkage between the terminus of the polymer backbone and thethioester group. The thioester-terminated polymers of the invention havethe structure:

[0067] wherein,

[0068] L is the point of bonding to a water soluble and non-peptidicpolymer backbone;

[0069] Z is a hydrolytically stable linkage or a hydrolytically unstablelinkage, such as O, S, —NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

[0070] m is from 0 to about 12, preferably 1 to about 4;

[0071] each X is independently selected from H and alkyl, such as C1-C6alkyl;

[0072] a is 0 or 1;

[0073] Y is a heteroatom, preferably O or S; and

[0074] Q is a sulfur-containing leaving group preferably having theformula —S—R₁, wherein R₁ is hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocycle, or substituted heterocycle.

[0075] A. Polymer Backbone

[0076] In general, the water soluble and non-peptidic polymer backboneshould be non-toxic and biocompatible, meaning that the polymer iscapable of coexistence with living tissues or organisms without causingharm. When referring to a thioester-terminated polymer backbone herein,it is to be understood that the polymer backbone can be any of a numberof water soluble and non-peptidic polymers, such as those describedbelow. Preferably, poly(ethylene glycol) (PEG) is the polymer backbone.The term PEG includes poly(ethylene glycol) in any of a number ofgeometries or forms, including linear forms (e.g., alkoxy PEG orbifunctional PEG), branched or multi-arm forms (e.g., forked PEG or PEGattached to a polyol core), pendant PEG, or PEG with degradable linkagestherein, to be more fully described below.

[0077] In its simplest form, PEG has the formula

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂  Formula II

[0078] wherein n is from about 10 to about 4000, typically from about 20to about 2000. Although the number average molecular weight of the PEGpolymer backbone can vary, PEGs having a number average molecular weightof from about 100 Da to about 100,000 Da, preferably about 5,000 Da toabout 60,000 Da are particularly useful. For example, PEG polymershaving a molecular weight of about 100 Da, about 200 Da, about 300 Da,about 500 Da, about 800 Da, about 1,000 Da, about 2,000 Da, about 3,000Da, about 4,000 Da, about 5,000 Da, about 10,000 Da, about 15,000, about20,000, about 30,000 and about 40,000 are useful in the presentinvention.

[0079] End-capped polymers, meaning polymers having at least oneterminus capped with a relatively inert group (e.g., an alkoxy group),can also be used as the polymer backbone of the invention. For example,methoxy-PEG-OH, or mPEG in brief, is a form of PEG wherein one terminusof the polymer backbone is bonded to a methoxy group, while the otherterminus is a hydroxyl group that is subject to ready chemicalmodification. The structure of mPEG is given below.

CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH  Formula III

[0080] wherein n is as described above.

[0081] Monomethoxy-terminated PEG molecules having a number averagemolecular weight of about 100 to about 100,000 Da, more preferably about2,000 to about 60,000 Da, are typically preferred for conjugating toproteins. Use of a monofunctional polymer such as mPEG preventscross-linking of the protein that often occurs when bifunctional ormultifunctional reagents are used. In the present invention,mPEG-thioester can be used to produce a single PEG molecule attached toa single protein molecule. However, in an alternate embodiment,utilizing a homobifunctional PEG-thioester in appropriate proportionswill result in a conjugate having two protein molecules attached to asingle PEG molecule, even in the event the protein contains multiplefree cysteine residues. Due to the manner in which the PEG derivative isbelieved to react (i.e. initially linking through the available thiolgroup of the N-terminal cysteine residue and then rearranging to formthe amide linkage), it is not possible for the thioester polymerderivatives of the invention to give a cross-linked protein becauseother free cysteine residues will not have both an available thiol groupand an available amine group. Thus, another advantage of the presentinvention is the ability to use polymers with multiple functional groupsof the type described herein without undesirable crosslinking with thepolypeptide.

[0082] Multi-armed or branched PEG molecules, such as those described inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety, can also be used as the PEG polymer. For example, the PEGpolymer backbone can have the structure:

[0083] wherein:

[0084] poly_(a) and poly_(b) are PEG backbones, such as methoxypoly(ethylene glycol);

[0085] R″ is a nonreactive moiety, such as H, methyl or a PEG backbone;and

[0086] P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

[0087] The PEG polymer may alternatively comprise a forked PEG. Anexample of a forked PEG is represented by PEG-YCHZ₂, where Y is alinking group and Z is an activated terminal group, such as the aldehydegroup of the present invention, linked to CH by a chain of atoms ofdefined length. International Application No. PCT/US99/05333, thecontents of which are incorporated by reference herein, disclosesvarious forked PEG structures capable of use in the present invention.The chain of atoms linking the Z functional groups to the branchingcarbon atom serve as a tethering group and may comprise, for example, analkyl chain, ether linkage, ester linkage, amide linkage, orcombinations thereof.

[0088] The PEG polymer may comprise a pendant PEG molecule havingreactive groups, such as carboxyl, covalently attached along the lengthof the PEG backbone rather than at the end of the PEG chain. The pendantreactive groups can be attached to the PEG backbone directly or througha linking moiety, such as an alkylene group.

[0089] In addition to the above-described forms of PEG, the polymer canalso be prepared with one or more weak or degradable linkages in thepolymer backbone, including any of the above described polymers. Forexample, PEG can be prepared with ester linkages in the polymer backbonethat are subject to hydrolysis. As shown below, this hydrolysis resultsin cleavage of the polymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

[0090] Other hydrolytically degradable linkages, useful as a degradablelinkage within a polymer backbone, include carbonate linkages; iminelinkages resulting, for example, from reaction of an amine and analdehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3(1997), which is incorporated herein by reference.); phosphate esterlinkages formed, for example, by reacting an alcohol with a phosphategroup; hydrazone linkages which are typically formed by reaction of ahydrazide and an aldehyde; acetal linkages that are typically formed byreaction between an aldehyde and an alcohol; ortho ester linkages thatare, for example, formed by reaction between a formate and an alcohol;peptide linkages formed by an amine group, e.g., at an end of a polymersuch as PEG, and a carboxyl group of a peptide; and oligonucleotidelinkages formed by, for example, a phosphoramidite group, e.g., at theend of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

[0091] It is understood by those skilled in the art that the termpoly(ethylene glycol) or PEG represents or includes all the above formsof PEG.

[0092] Many other polymers are also suitable for the invention. Any of avariety of monofunctional, bifunctional or multifunctional polymerbackbones that are non-peptidic and water-soluble could be used in thepresent invention. The polymer backbone can be linear, or may be in anyof the above-described forms (e.g., branched, forked, and the like).Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), copolymers of ethylene glycol and propyleneglycol, poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such asdescribed in U.S. Pat. No. 5,629,384, which is incorporated by referenceherein in its entirety, and copolymers, terpolymers, and mixturesthereof.

[0093] B. Linkage Between Polymer Backbone and Thioester

[0094] The intervening linkage between the terminus of the polymerbackbone and the thioester group is the residue of the functional groupon the polymer backbone that couples the polymer backbone to theterminal thioester group. Thus, as would be understood, the structure ofthe linkage will vary depending on the structure of the functional groupof the polymer backbone. The linkage can comprise a hydrolyticallystable linkage, such as amide, urethane, ether, thioether, or urea.Alternatively, the linkage can comprise a hydrolytically unstablelinkage, such as carboxylate ester, phosphate ester, orthoester,anhydride, imine, acetal, ketal, oligonucleotide, or peptide. In oneembodiment, in addition to the hydrolytically stable or unstablelinkage, the linkage between the polymer backbone and the thioesterincludes an optional alkylene spacer, designated herein as (CHX)_(m).

[0095] As shown above in Formula I, the linkage preferably has thestructure:

[0096] wherein:

[0097] Z is the hydrolytically stable or unstable linkage, such as O, S,—NHCO—, —CONH—, —O₂C—, —NHCO₂—, or —O₂CNH—;

[0098] m is from 0 to about 12, preferably 1 to about 4;

[0099] each X is independently selected from H and alkyl, such as C1-C6alkyl; and

[0100] a is 0 or 1.

[0101] The length of the alkylene chain (i.e., the value of in) can varyfrom 0 to about 12. For example, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12. Preferably, m is 0, 1, 2, 3, or 4. Each X of the alkylenechain is preferably hydrogen, methyl or ethyl. In a preferredembodiment, a is 1 and Z is a heteroatom, such as O or S.

[0102] C. Thioester Functional Group

[0103] The thioester functional group is covalently attached to at leastone terminus of the water soluble polymer. The thioester group has thestructure:

[0104] wherein:

[0105] Y is a heteroatom, preferably O or S; and

[0106] Q is a sulfur-containing electrophilic leaving group preferablyhaving the formula —S—R₁, wherein R₁ is hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocycle, or substitutedheterocycle.

[0107] The particular R₁ group employed can vary. The R₁ group, inconjunction with the sulfur atom, forms an electrophilic leaving groupsuitable for displacement during nucleophilic attack of the carbonylcarbon by the thiol or imidazole side chain of the N-terminal amino acidresidue of a polypeptide. Preferred R₁ groups include substituentsderived from phenol, nitrophenol, benzoic acid, pyridine,pyridinecarboxylic acid, and nitropyridine. Substituted or unsubstitutedpyridinyl is particularly preferred. Examples 1-3 illustratethioester-terminated PEG polymers bearing a thiopyridinyl leaving group.

[0108] D. Exemplary Polymer Structures

[0109] An embodiment of a linear polymer of the invention can bestructurally represented as shown below:

[0110] wherein POLY is a water soluble and non-peptidic polymerbackbone, R is a capping group or a functional group, and Z, X, Y, m, aand Q are as defined above. In a preferred embodiment, R is methoxy,POLY is poly(ethylene glycol), a is 1, Z is O, m is 1 to about 3, Y isO, and each X is H or CH₃.

[0111] The R group can be a relatively inert capping group, such asalkoxy (e.g., methoxy or ethoxy), alkyl, benzyl, aryl, or aryloxy (e.g.,benzyloxy). Alternatively, the R group can be a functional group capableof readily reacting with a functional group on a biologically activemolecule. Exemplary functional groups include hydroxyl, active ester(e.g. N-hydroxysuccinimidyl ester or 1-benzotriazolyl ester), activecarbonate (e.g. N-hydroxysuccinimidyl carbonate and 1-benzotriazolylcarbonate), acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, hydrazide, thiol,carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione,mesylate, tosylate, or tresylate. Specific examples of terminalfunctional groups for the polymer backbones of the invention includeN-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zalipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al., Makromol.Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references are incorporatedherein by reference.

[0112] In a homobifunctional embodiment of Formula V, R is athioester-containing moiety of formula —(Z)_(a)—(CXH)_(m)—CO—S—R₁,wherein Z, a, x, m, and R₁ are as defined above.

[0113] Some specific examples of linear polymers of the invention areshown below:

[0114] wherein R₁ and n are as defined above.

[0115] One example of a multi-arm embodiment of the thioester-terminatedpolymer of the invention has the structure:

[0116] wherein each POLY is a water soluble and non-peptidic polymerbackbone, R′ is a central core molecule, y is from about 3 to about 100,preferably 3 to about 25, and Z, X, Y, m, a and R₁ are as defined above.The core moiety, R′, is a residue of a molecule selected from the groupconsisting of polyols, polyamines, and molecules having a combination ofalcohol and amine groups. Specific examples of central core moleculesinclude glycerol, glycerol oligomers, pentaerythritol, sorbitol, andlysine.

[0117] The central core molecule is preferably a residue of a polyolhaving at least three hydroxyl groups available for polymer attachment.A “polyol” is a molecule comprising a plurality of available hydroxylgroups. Depending on the desired number of polymer arms, the polyol willtypically comprise 3 to about 25 hydroxyl groups. The polyol may includeother protected or unprotected functional groups as well withoutdeparting from the invention. Although the spacing between hydroxylgroups will vary from polyol to polyol, there are typically 1 to about20 atoms, such as carbon atoms, between each hydroxyl group, preferably1 to about 5. Preferred polyols include glycerol, reducing sugars suchas sorbitol, pentaerythritol, and glycerol oligomers, such ashexaglycerol. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.The particular polyol chosen will depend on the desired number ofhydroxyl groups needed for attachment to the polymer arms.

[0118] E. Method of Forming Thioester Polymers

[0119] The thioester polymers of the invention may be formed byderivatization of a water-soluble non-peptidic polymer by any of anumber of synthetic approaches for forming thioesters known in the art.See, for example, Field, L. Synthesis, 1972, 106. For instance, athioester can be prepared from the corresponding acidchloride-terminated polymer by reaction with a thallium(I) salt of athiolate (Spessard, G., et al., Organic Synthesis Collection, Vol. 7,87). For thioester derivatization of a polymer having additionalfunctional groups contained within the molecule, such as hydroxy orother functional groups, alternative approaches such as the followingmay be employed. For example, a thioester-terminated polymer asdescribed herein can be formed from the corresponding carboxylicacid-terminated polymer by reaction of the acid with a dialkyl ordiphenyl phosphorochloridate to form the anhydride, which can then beconverted to the corresponding thioester. (Masamune, S., et al., Can. J.Chem., 1975, 53, 3693; Yamada, S., et al., Chem. Pharm. Bull. 1977, 25,2423). In yet another synthetic approach, a thioester-terminated polymercan be prepared by reaction of an imidazolide of a carboxylic acid(prepared by reaction of the corresponding carboxylic acid withN,N-carbonyldiimidazole) with a relatively acidic thiol (Masamune, S.,et al., J. Am. Chem. Soc., 1976, 98, 7874). Alternatively, a disulfideand triphenylphosphine can be used to convert a carboxylic acid terminusof a polymer to the corresponding thioester (Mukaiyama, T., et al.,Bull. Chem. Soc. Jpn., 1970, 43, 1271). Other methods that can be usedto prepare thioesters from carboxylic acids include the use of arylthiocyanates (Grieco, P., et al., J. Org. Chem., 1978, 43, 1283),thiopyridyl chloroformate (Corey, E. J., et al., Tetrahedron Lett.,1979, 2875), 2-fluoro-N-methylpyridinium tosylate (Watanabe, Y., et al.,Chem. Lett. 1976, 741), 1-hydroxybenzotrizaole (Horiki, K., Synth.Commun. 1977, 7, 251), and boron thiolate (Pelter, A., et al., J. Chem.Soc., Perkin Trans. I, 1977, 1672). Alternatively, a polymer having anO-ester terminus can be converted to the corresponding S-ester byaluminum and boron reagents.

[0120] A preferred method of forming the thioester polymers of theinvention involves base-catalyzed reaction of a terminal carboxylicacid, or active ester thereof, of a reactive polymer with a thiolcompound of formula R₁—SH, wherein R₁ is as defined above. Preferredreactive polymers bearing a terminal carboxylic acid group includepoly(ethylene glycol) terminated with a carboxymethyl, propionic acid,or butanoic acid group. Any other method known in the art for coupling athioester group to a terminus of a polymer backbone, such as any ofthose described above, could also be used without departing from thepresent invention. Exemplary methods of forming thioester-terminatedpolymers are illustrated in Examples 1-3.

[0121] III. Polymer/Polypeptide Conjugates

[0122] A. Structure of Polymer/Polypeptide Conjugate

[0123] The thioester polymers of the invention selectively react withthe α-amine of a polypeptide having a histidine or cysteine molecule atthe N-terminus to form an amide linkage between the polymer and thepolypeptide. In a preferred embodiment, the polymer-polypeptideconjugate comprises a water soluble and non-peptidic polymer backbonehaving at least one terminus bonded to the structure:

[0124] wherein:

[0125] L, Z, Y, m, X and a are defined above;

[0126] W is —CH₂SH or

[0127]  depending on whether the terminal amino acid is cysteine orhistidine; and

[0128] POLYPEPTIDE is the polypeptide molecule. The polymer backbone cancomprise any of the polymer structures discussed above, such as PEG inany of its forms.

[0129] The polypeptide can be any polypeptide having an N-terminalcysteine or histidine residue, regardless of whether the N-terminalcysteine or histidine is naturally occurring in the polypeptide orintroduced by modification of the polypeptide sequence. The polypeptidemolecule is preferably selected from the group consisting of proteins,protein-ligands, enzymes, cytokines, hematopoietins, growth factors,hormones, antigens, antibodies, antibody fragments, receptors, andprotein fragments. The following is an illustrative although by no meansexhaustive list of polypeptide molecules that include, or could bemodified to include, an N-terminal cysteine or histidine residue:calcitonin, parathyroid hormone, interferon alpha, interferon beta,interferon gamma, interleukins 1-21, granulocyte-colony stimulatingfactor, macrophage-colony stimulating factor, granulocyte-macrophagecolony stimulating factor, stem cell factor, leukemia inhibitory factor,kit-ligand, flt-3 ligand, erythropoietin, thrombopoietin, tumor necrosisfactor alpha, tumor necrosis factor beta, transforming growth factor,bone morphogenic proteins, osteoprotegerin, tissue plasminogenactivator, platelet derived growth factor, fibroblast growth factor,keratinocyte growth factor, epidermal growth factor, human growthhormone, insulin, tumor necrosis factor-related apoptosis-inducingligand (TRAIL), DNAse, receptors, enzymes, fusion proteins, chimericantibodies, humanized antibodies, fully human antibodies, Fab fragments,F(ab′)₂ fragments, Fv fragments, and scFv fragments. In one preferredembodiment, the polypeptide is an interferon molecule.

[0130] An exemplary embodiment of a linear polymer conjugate of theinvention has the structure:

[0131] wherein R, POLY, Z, a, X, m, Y and W are as defined above.

[0132] In an alternative embodiment where the polymer is a multi-armpolymer, an exemplary polymer conjugate of the invention has thestructure:

[0133] wherein R′, y, POLY, Z, a, X, m, Y and W are as defined above.

[0134] Polypeptide conjugates in accordance with the invention willpossess an amide linkage formed by reaction with an N-terminal cysteineor histidine of the polypeptide, where the polymer portion of theconjugate may have any of a number of different geometries (e.g.,linear, branched, forked, and the like), molecular weights, optionaldegradable linkages, etc., as described in detail herein and in theaccompanying examples. Representative conjugates prepared in accordancewith the invention are provided in Examples 4-7.

[0135] B. Method of Forming Polymer/Polypeptide Conjugate

[0136] The present invention uses a thioester-terminated polymer, suchas a thioester-terminated PEG, to specifically modify the a-amine of anN-terminal cysteine or histidine without permanently modifying theremaining free functional group (e.g., the thiol group of a cysteineresidue) on the terminal amino acid or modifying other amine groupspresent in the polypeptide chain. Although not bound by any particulartheory, Reaction Scheme I below illustrates the reaction believed tooccur between a polypeptide having an N-terminal cysteine molecule and areactive polymer of the invention. As shown, it is believed that thethioester-terminated polymer initially reacts with the free thiol groupof the cysteine and thereafter undergoes an intramolecular rearrangementto form an amide linkage with the N-terminal amine group, thus leavingthe thiol group available for further modification if desired. Thethiol-thioester exchange is preferably effected by use of atrialkylphosphine, such as tris(2-carboxyethyl)phosphine ortriethylphosphine, and optionally a thiol, such as mercaptopropionicacid.

[0137] Optionally, in the case of an N-terminal cysteine molecule, asecond thiol-reactive polymer (e.g., a thiol-reactive PEG) may bereacted with the free thiol group in order to form a branched structureat the N-terminus of the polypeptide as shown in Reaction Scheme I,wherein L′ is the linker resulting from the reaction of thethiol-reactive terminal functional group of the second PEG polymer withthe free thiol group on the cysteine molecule. In one embodiment, onlytwo polymer backbones are attached to the polypeptide.

[0138] Examples of thiol-reactive functional groups includevinylsulfone, maleimide, orthopyridyl disulfide and iodoacetamide.Examples of the L′ linkage include:

[0139] (resulting from vinylsulfone functional group)

[0140] (resulting from maleimide functional group)

[0141] —S—S—

[0142] (resulting from orthopyridyl disulfide functional group)

[0143] (resulting from iodoacetamide functional group)

[0144] As would be readily understood by one of ordinary skill in theart, the method of the invention could be used to couple theabove-described polymer derivatives to any moiety, whether peptidic ornot, having a terminal —CH(W)—NH₂ group, wherein W is as defined above.

IV. EXAMPLES

[0145] The following examples are given to illustrate the invention, butshould not be considered in limitation of the invention. For example,although MPEG is used in the examples to illustrate the invention, otherforms of PEG and similar polymers that are useful in the practice of theinvention are encompassed by the invention as discussed above.

[0146] All PEG reagents referred to in the appended examples areavailable from Shearwater Corporation of Huntsville, Ala. All ⁺HNMR datawas generated by a 300 or 400 MHz NMR spectrometer manufactured byBruker.

[0147] Examples 1-3 illustrate methods of forming a thioester-terminatedpolymer of the invention. Examples 4-7 illustrate reaction of athioester-terminated polymer of the invention with an exemplarypolypeptide having a N-terminal cysteine residue. As indicated below,use of the thioester polymers of the invention results in selectiveattachment of the polymer to the N-terminal amine of the polypeptide.

Example 1 Preparation of PEG(5000)-α-methoxy-ω-propionic Acid,2-pyridylthioester (PEG-PA-OPTE)

[0148]

[0149] 2-mercaptopyridine (40.0 mg, 0.36 mmoles), 1-hydroxybenzotriazole(4.0 mg, 0.030 mmoles), 4-(dimethylamino)pyridine (36.7 mg, 0.30 mmoles)and 1,3-dicyclohexylcarbodiimide (dissolved in 2 mL anhydrousdichloromethane, 84.0 mg, 0.41 mmoles) were added to a solution ofPEG(5000)-α-methoxy-ω-propionic acid (1.5 g, 0.27 mmoles) in anhydrousacetonitrile (20 mL). The reaction solution was stirred overnight atambient temperature under argon. The solution was then concentrated tonear dryness at reduced pressure, followed by addition of anhydroustoluene (50 mL). The mixture was stirred at room temperature for thirtyminutes, filtered and the filtrate was concentrated at reduced pressureto near dryness. Ethyl acetate (200 mL) was added and the mixture waswarmed until the contents were completely dissolved. The solution wasthen cooled to room temperature while stirring. Ethyl ether (50 mL) wasadded and a precipitate formed. The product was filtered and rinsed withethyl ether until the product became white. The product was then driedunder high vacuum. Yield: 1.1 g. NMR (d6-DMSO): 62.98 ppm (t, 2H,—CH₂—COS—), δ3.51 ppm (s, PEG backbone), δ7.46 ppm (m, ill resolved, 1H,H₅ (pyridyl)), δ6.64 ppm (d, 1H, H₃ (pyridyl)), δ7.91 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 2 Preparation of PEG(5000)-α-benzyloxy(BZO)-ω-carboxymethyl,2-pyridylthioester (PEG-CM-OPTE)

[0150]

[0151] 2-mercaptopyridine (40.0 mg, 0.36 mmoles), 1-hydroxybenzotriazole(5.0 mg, 0.035 mmoles), and 1,3-dicyclohexylcarbodiimide (dissolved in 2mL anhydrous dichloromethane, 74.3 mg, 0.36 mmoles) were added to asolution of PEG(5000)-α-benzyloxy-ω-carboxymethyl (1.5 g, 0.30 mmoles)in anhydrous acetonitrile (20 mL). The reaction solution was stirredovernight at ambient temperature under argon. The solution was thenconcentrated to near dryness at reduced pressure, followed by additionof anhydrous toluene (30 mL). The mixture was stirred at roomtemperature for thirty minutes, filtered and the filtrate wasconcentrated at reduced pressure to near dryness. Ethyl acetate (150 mL)was added and the mixture was warmed until the contents were completelydissolved. The solution was then cooled to room temperature whilestirring. Ethyl ether (50 mL) was added to the solution and aprecipitate formed. The product was filtered and rinsed with ethyl etheruntil the product became white. The product was then dried under highvacuum. Yield: 1.1 g. NMR (d6-DMSO): δ3.51 ppm (s, PEG backbone), δ4.39ppm (s, 2H, —OCH₂COS—), δ4.49 ppm (s, 2H, —OCH₂-(benzyloxy)), δ7.33 ppm(m, ill resolved, 5H, C₆H₅ (benzyloxy)), δ7.46 ppm (m, ill resolved, 1H,H₅ (pyridyl)), δ7.63 ppm (d, 1H, H₃ (pyridyl)), δ7.91 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 3 Preparation of PEG(5000)-α-methoxy-ω-2-methyl butanoic acid,2-pyridylthioester

[0152]

[0153] 2-mercaptopyridine (44.5 mg, 0.40 mmoles), 1-hydroxybenzotriazole(4.7 mg, 0.033 mmoles), 4-(dimethylamino)pyridine (40.7 mg, 0.33 mmoles)and 1,3-dicyclohexylcarbodiimide (dissolved in 2 mL anhydrousdichloromethane, 92.8 mg, 0.45 mmoles) were added to a solution ofPEG(5000)-α-methoxy-ω-2-methyl butanoic acid (1.5 g, 0.30 mmoles) inanhydrous acetonitrile (20 mL). The reaction solution was stirredovernight at ambient temperature under argon. The solution was thenconcentrated to near dryness at reduced pressure, followed by additionof anhydrous toluene (50 mL). The mixture was stirred at roomtemperature for thirty minutes, filtered and the filtrate wasconcentrated at reduced pressure to near dryness. Ethyl acetate (150 mL)was added and mixture was warmed until the contents completelydissolved. The solution was then cooled to room temperature whilestirring. A precipitate was formed by adding 2-Propanol (50 mL),followed by addition of ethyl ether (50 mL). The product was filteredoff, rinsed with 2-propanol until the product became white. The productwas then dried under high vacuum. Yield: 1.2 g. NMR (d6-DMSO): δ1.19 ppm(d, 3H, —O—CH₂—CH₂—CH(CH₃)—COS—), δ1.66 ppm and δ1.92 ppm (mm, 2H,—O—CH₂—CH₂—CH(CH₃)—COS—), δ2.89 ppm (m, 1H, —O—CH₂—CH₂—CH(CH₃)—COS—),δ3.51 ppm (s, PEG backbone), δ7.46 ppm (m, ill resolved, 1H, H₅(pyridyl)), δ7.63 ppm (d, 1H, H₃ (pyridyl)), δ7.90 ppm (t, 1H, H₄(pyridyl)), δ8.60 ppm (d, 1H, H₆ (pyridyl)).

Example 4 Conjugation of PEG-CM-OPTE to Interferon

[0154] Interferon tau (0.45 mg), which has a cysteine as the N-terminalamino acid, was formulated to 0.3 mg/ml in 1M Tris, 0.7 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 3 mM mercaptopropionicacid at pH 7.75. Approximately 1.0 mg of mPEG_(5K)-CM-OPTE (from Example2) was added to the interferon solution and allowed to react at roomtemperature for 4 hours. The reaction mixture was dialyzed againstdeionized water overnight. The product was analyzed by MALDI-MS. Themass spectrum showed free PEG at 5000 Da, unconjugated interferon at19,979 Da and a single PEG conjugate at a molecular weight of 25,065 Da,meaning the PEGylated product has only a single PEG molecule attached tothe polypeptide at the N-terminus.

Example 5 Conjugation of PEG-PA-OPTE to Interferon

[0155] Interferon tau (0.45 mg) was formulated to 0.3 mg/ml in 0.33MTris, 0.7 mM TCEP (Tris[2-carboxyethylphosphine]hydrochloride) at pH7.75. Approximately 1.0 mg of mPEG_(5K)-PA-OPTE (orthopyridyl thioesterof propionic acid from Example 1) was added to the interferon solutionand allowed to react at room temperature for 4 hours. The product wasanalyzed by SDS-PAGE. The gel showed two bands corresponding tounconjugated interferon (˜20 kDa) and singly PEG-conjugated interferon(˜29 kDa) (i.e., a polypeptide attached to a single PEG molecule). Theslower migration of the PEG-interferon conjugate is due to the largerhydrodynamic volume of the PEG chain when compared to a correspondingmolecular weight protein.

Example 6 Conjugation of PEG-CM-OPTE to a Polypeptide

[0156] The polypeptide CRASKSVSSSGYSYMHWYQQ (MW=2355 Da) (SEQ ID NO: 1)was formulated to 0.67 mg/ml in 0.67M Tris, 1.3 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 5.3M urea at pH 7.75.Approximately 21.0 mg of mPEG₅K-CM-OPTE (from Example 2) was added tothe polypeptide solution and allowed to react at room temperature for 4hours. The reaction mixture was dialyzed against deionized waterovernight. The product was analyzed by MALDI-MS. The mass spectrumshowed a conjugate comprising a single PEG molecule attached to thepolypeptide and having a molecular weight of 7555 Da. This demonstratesthat the thioester-terminated polymer did not randomly react with otherfree amine groups in the molecule, such as the amine groups of thelysine or arginine residues.

Example 7 Conjugation of PEG-PA-OPTE to a Polypeptide

[0157] The polypeptide CRASKSVSSSGYSYMHWYQQ (MW=2355 Da) (SEQ ID NO: 1)was formulated to 0.67 mg/ml in 0.67M Tris, 1.3 mM TCEP(Tris[2-carboxyethylphosphine]hydrochloride) and 5.3M urea at pH 7.75.Approximately 21.0 mg of mPEG₅K-PA-OPTE (from Example 1) was added tothe polypeptide solution and allowed to react at room temperature for 4hours.

[0158] Many modifications and other embodiments of the invention willcome to mind to one skilled in the art to which this invention pertainshaving the benefit of the teachings presented in the foregoingdescription. Therefore, it is to be understood that the invention is notto be limited to the specific embodiments disclosed and thatmodifications and other embodiments are intended to be included withinthe scope of the appended claims. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

1 1 1 20 PRT Artificial Sequence Polypeptide having 20 amino acidresidues and an N-terminal cysteine. 1 Cys Arg Ala Ser Lys Ser Val SerSer Ser Gly Tyr Ser Tyr Met His 1 5 10 15 Trp Tyr Gln Gln 20

That which is claimed:
 1. A thioester-terminated reactive polymer,comprising a water soluble and non-peptidic polymer backbone having atleast one terminus bonded to the structure:

wherein: L is the point of bonding to the polymer backbone; Z is alinker; m is from 0 to about 12; Y is a heteroatom; each X isindependently selected from H and alkyl; a is 0 or 1; and Q is asulfur-containing leaving group.
 2. The reactive polymer of claim 1,wherein each X is H or C1-C6 alkyl.
 3. The reactive polymer of claim 2,wherein each X is H or methyl.
 4. The reactive polymer of claim 1,wherein Y is O or S.
 5. The reactive polymer of claim 1, wherein Q hasthe formula —S—R₁, wherein R₁ is selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,and substituted heterocycle.
 6. The reactive polymer of claim 5, whereinR₁ is selected from the group consisting of phenol, nitrophenol, benzoicacid, pyridine, pyridinecarboxylic acid, and nitropyridine.
 7. Thereactive polymer of claim 5, wherein R₁ is substituted or unsubstitutedpyridine.
 8. The reactive polymer of claim 1, wherein a is 1 and Z isselected from the group consisting of —O—, —S—, —NHCO—, —CONH—, —O₂C—,—NHCO₂—, and —O₂CNH—.
 9. The reactive polymer of claim 1, wherein thepolymer backbone is selected from the group consisting of poly(alkyleneglycol), poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),polyacrylate, polyacrylamides, polysaccharides, and copolymers,terpolymers, and mixtures thereof.
 10. The reactive polymer of claim 1,wherein said water soluble and non-peptidic polymer backbone is selectedfrom the group consisting of poly(ethylene glycol), poly(propyleneglycol), and copolymers of ethylene glycol and propylene glycol.
 11. Thereactive polymer of claim 1, wherein said polymer backbone ispoly(ethylene glycol) having a number average molecular weight of about100 Da to about 100,000 Da.
 12. The reactive polymer of claim 1, whereinm is 1 to about
 4. 13. The reactive polymer of claim 1, having thestructure:

wherein: POLY is a water soluble and non-peptidic polymer backbone; R isa capping group or a functional group; and R₁ is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocycle, and substituted heterocycle.
 14. The reactivepolymer of claim 13, wherein R is selected from the group consisting ofalkoxy, alkyl, benzyl, aryl, aryloxy, hydroxyl, active ester, activecarbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, hydrazide, thiol,carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione,mesylate, tosylate, tresylate and —(Z)_(a)—(CXH)_(m)—CO—S—R₁.
 15. Thereactive polymer of claim 13, wherein POLY is poly(ethylene glycol). 16.The reactive polymer of claim 1, having the structure:

wherein: each POLY is a water soluble and non-peptidic polymer backbone;R′ is a central core molecule; y is from about 3 to about 100; and R₁ isselected from the group consisting of hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocycle, and substitutedheterocycle.
 17. The reactive polymer of claim 16, wherein POLY ispoly(ethylene glycol).
 18. The reactive polymer of claim 16, wherein R′is a residue of a molecule selected from the group consisting ofpolyols, polyamines, and molecules having a combination of alcohol andamine groups.
 19. The reactive polymer of claim 16, wherein R′ is aresidue of a molecule selected from the group consisting of glycerol,glycerol oligomers, pentaerythritol, sorbitol, and lysine.
 20. A polymerconjugate of a polypeptide having a cysteine or histidine residue at theN-terminus, said polymer conjugate comprising a water soluble andnon-peptidic polymer backbone having at least one terminus bonded to thestructure:

wherein: L is the point of bonding to the polymer backbone; Z is alinker; Y is a heteroatom; m is from 0 to about 12; each X isindependently selected from H and alkyl; a is 0 or 1; W is —CH₂SH or

 and POLYPEPTIDE is the polypeptide molecule.
 21. The polymer conjugateof claim 20, wherein the polymer backbone is poly(ethylene glycol). 22.The polymer conjugate of claim 20, wherein POLYPEPTIDE is selected fromthe group consisting of proteins, protein-ligands, enzymes, cytokines,hematopoietins, growth factors, hormones, antigens, antibodies, antibodyfragments, receptors, and protein fragments.
 23. The polymer conjugateof claim 20, wherein POLYPEPTIDE is an interferon molecule.
 24. A methodof conjugating a polymer derivative to a polypeptide having a cysteineor histidine residue at the N-terminus, said method comprising:providing a polypeptide having a cysteine or histidine residue at theN-terminus; providing a thioester-terminated polymer, the polymercomprising a water soluble and non-peptidic polymer backbone having atleast one terminus bonded to the structure:

wherein: L is the point of bonding to the polymer backbone; Z is alinker; m is from 0 to about 12; Y is a heteroatom; each X isindependently selected from H and alkyl; a is 0 or 1; and Q is asulfur-containing leaving group; reacting the thioester-terminatedpolymer with the polypeptide to form a conjugate having the structure:

wherein POLYPEPTIDE is the polypeptide molecule, and W is —CH₂SH or


25. The method of claim 24, wherein the polymer backbone ispoly(ethylene glycol).
 26. The method of claim 24, wherein POLYPEPTIDEis selected from the group consisting of protein ligands, enzymes,cytokines, hematopoietins, growth factors, hormones, antigens,antibodies, antibody fragments, receptors, and protein fragments.
 27. Apolymer conjugate of a polypeptide having a cysteine molecule at theN-terminus, said polymer conjugate comprising two water soluble andnon-peptidic polymer backbones attached at the N-terminus, the conjugatehaving the structure:

wherein: L is the point of bonding to each of said two polymerbackbones, L′ and Z are linkers, Y is a heteroatom; m is from 0 to about12; each X is independently selected from H and alkyl; a is 0 or 1; andPOLYPEPTIDE is the polypeptide molecule.
 28. The polymer conjugate ofclaim 27, wherein L′ is selected from the group consisting of